Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Inhibitory evaluation of Curculigo latifolia on

α-glucosidase, DPP (IV) and in vitro studies in
antidiabetic with molecular docking relevance to
type 2 diabetes mellitus

Nur Athirah Zabidi, Nur Akmal Ishak, Muhajir Hamid, Siti Efliza Ashari &
Muhammad Alif Mohammad Latif

To cite this article: Nur Athirah Zabidi, Nur Akmal Ishak, Muhajir Hamid, Siti Efliza Ashari
& Muhammad Alif Mohammad Latif (2021) Inhibitory evaluation of Curculigo latifolia on α-
glucosidase, DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to
type 2 diabetes mellitus, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 109-121,
DOI: 10.1080/14756366.2020.1844680

To link to this article: https://doi.org/10.1080/14756366.2020.1844680

© 2020 The Author(s). Published by Informa Published online: 29 Nov 2020.

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 109–121
https://doi.org/10.1080/14756366.2020.1844680

RESEARCH PAPER

Inhibitory evaluation of Curculigo latifolia on a-glucosidase, DPP (IV) and in vitro

studies in antidiabetic with molecular docking relevance to type 2
diabetes mellitus
Nur Athirah Zabidia, Nur Akmal Ishaka,b, Muhajir Hamidc, Siti Efliza Asharib,d and
Muhammad Alif Mohammad Latifb,d
a
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; bCentre of Foundation
Studies for Agricultural Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; cDepartment of Microbiology, Faculty of Biotechnology
and Molecular Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; dIntegrated Chemical Biophysics Research, Faculty of Science,
Universiti Putra Malaysia, Serdang, Selangor, Malaysia

ABSTRACT ARTICLE HISTORY

The inhibition of a-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in Received 5 June 2020
the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory poten- Revised 13 October 2020
tial of a-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion Accepted 27 October 2020
activities through in vitro studies. The selected of active compounds obtained from the screening of com-
KEYWORDS
pounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From a-glucosidase; DPP (IV);
the results, root extracts displayed a better promising outcome in a-glucosidase (IC50 2.72 ± 0.32) as com- Curculigo latifolia; molecular
pared with the fruit extracts (IC50 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the docking; type 2 diabetes
inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results
revealing that phlorizin binds strongly with a-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with
achieving the lowest binding energy value. The present work suggests several of the compounds have
the potential that contribute towards inhibiting a-glucosidase and DPP (IV) and thus effective in lowering
post-prandial hyperglycaemia.

Introduction digestion and increased in peripheral glucose uptake5. One of the

Diabetes mellitus (DM) is a chronic carbohydrate metabolism dis-
order that has an abnormal homeostasis of glucose leading to a
high blood glucose levels.Globally, diabetes mellitus (DM) is recog-
nised as one of the major disease in the world with the highest
crisis diseases in the 21st century1. According to the International
Diabetes Federation in the 9th edition, it has been revealed that
approximately 425 million adults between ages 20–79 years were
diagnosed with diabetes worldwide in 2019. However, if this does
not recover or reduced, this figure is expected to rise around 700
million by 2045 and based on records, almost 49.7% was undiag-
nosed and living without knowing they had this disease2.
Globally, based on the records, 374 million of people is at risk due
to the developing in type 2 diabetes mellitus (T2DM), hence these
evolution is increasing awareness of people about T2DM3.
Type 2 diabetes mellitus or shortly known as T2DM is a type of
diabetes disease that has features of insulin resistance from the
decrease in the peripheral glucose uptake in most of the targeted
tissue. This is happen due to the insensitivity of insulin from the
insulin resistance that may reduce the production of insulin and
finally leading to excessive blood glucose level or known as
hyperglycaemia4. Recently, the pathogenies of T2DM is inter-
twined with various type of mechanism such as increasing insulin
secretion, insulin sensitivity, increased response towards incretin
hormones, decrease reabsorption of glucose through carbohydrate
most potent approaches in the management of T2DM is by
Dipeptidyl peptidase (IV) inhibition, where the inhibitor of these
enzymes will prevent the degradation of the incretin hormone,
which in turn increase the insulin secretion and lowering blood
glucose level6. Apart from this, the excessive amount of carbohy-
drates present in the diet can be considered as one of the major
causes of diabetes. The delaying absorption of sugar in the blood-
stream that may exert in low blood sugar level which through an
inhibitory potential of a-glucosidase, known as the main enzyme
in the carbohydrate mechanism. The inhibition of these enzymes
is able to delay the absorption of monosaccharides from dietary
complex carbohydrates, preventing any sudden rise in meal-
induced blood glucose level, and therefore are involved in con-
trolling the glucose level in the blood7. This is well correlated with
the interesting finding that reveals that the a-glucosidase enzyme
inhibitor is capable of slowing carbohydrate digestion, resulting in
less efficient glucose absorption and thus decreasing postprandial
hyperglycaemia8.
Treatment in T2DM is mostly by consuming the oral antidia-
betic drugs that act through the different mechanisms in control-
ling the increased blood glucose level along with changes in diet
and healthy lifestyles. Clinically, there is various type of oral hyper-
glycaemic drugs, which has been used in controlling this disease
and are classified into different classes such as biguanides, sulfo-
nylureas, thiazolidinediones (TZD), meglitinides, Dipeptidyl

CONTACT Nur Akmal Ishak nur_akmal@upm.edu.my Centre of Foundation studies for Agricultural Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
110 N. A. ZABIDI ET AL.
peptidase (IV) inhibitors, sodium-glucose cotransporter (SGLT2),
and a-glucosidase inhibitors9. Each of the classes is targeting a dif-
ferent type of organ and are carried out in the various mode of
action. In fact, usually, combinations of different antidiabetic drugs
are used to increase the efficacy of the treatment10. However, des-
pite its benefits, these drugs also have their own long-term effects
that can give out adverse effects caused by chronic administra-
tion, including cardiovascular disease, lactate acidosis, hypogly-
caemia, gastrointestinal complaints and others11. To date,
molecular docking studies have been used recently in predicting
the potential inhibitor that has been compared with marketed
anti-diabetic drugs as which by seeking these natural compounds
as new anti-diabetic agents with little or no adverse effects for
the long term effect. Molecular docking is an important computa-
tional method for predicting possible drug-protein interactions
with the selected ligand that represent bioactive compound con-
taining in the medicinal plants12.
Nowadays, a new approach is introduced by applying plant
extracts, which may contain high therapeutic values as an alterna-
tive way of mimicking the function of antidiabetic drugs and also
have been reported to contain anti-diabetic properties13. Likewise,
Curculigo latifolia Dryand. ex W.T.Aiton, or locally known as lemba
is a rhizome geophyte that is categorised under family
Hypoxidaceae. From the previous study, C. latifolia has been
revealed has the ability to exhibit antidiabetic properties through
cell lines and in diabetic induced rats14. The root extract of C. lati-
folia has shown to modulate glucose and lipid metabolism in
experimental diabetic rats and the presence of curculigoside may
have bestowed its anti-diabetic efficacy14,15. Nonetheless, there
were no previous studies have yet been revealed on the inhibition
of DPP (IV), a-glucosidase assays, and molecular docking and this
present research could be regarded as an additional confirmation
of antidiabetic properties for this crop. The C latifolia root extracts
were previously screened for total phenolic content (TPC), total
flavonoid content (TFC), antioxidant activity and screening of
active compounds through LCMS analysis16. Therefore, the current
studies are conducted with an objective to evaluate antidiabetic
properties in the C. latifolia root and fruits extracts with the use of
environmentally extraction’s technique, which is subcritical water
extraction (SWE) method through inhibitory activities against
a-glucosidase and DPP (IV) enzyme along with in vitro studies by
examining the glucose uptake and insulin secretion. Additionally,
molecular docking simulation was also been performed in order
to predict the molecular docking scores by identifying the docking
score in each targeted protein involved.
Materials and methods
Plant materials
The C. latifolia plant was collected in Selangor, Malaysia.
(Geographical Coordinates: 2.8833
N, 101.8667
E). A voucher spe-
cimen of the plant (SK 1709/09) was confirmed and deposited in
the Biodiversity Unit, Institute of Bioscience, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia. The fruits are plucked and
cleaned with the tap water blotted with tissue paper and stored
at 20
C for overnight and freeze-dried. Roots are cleaned with
tap water and subsequently dried overnight in an oven at 40
C.
The dried roots and fruits were ground into a fine powder that
passed through a 30-mesh sieve. The powdered roots and fruits
were sealed and kept at 4
C for future use.
Materials and reagents
Methanol, acetonitrile, formic acid and ethanol (HPLC grade) were
purchased from Merck (Darmstadt, Germany). All other chemicals
were of analytical grade purity. All aqueous solutions were pre-
pared using ultrapure water. Drugs (acarbose, sitagliptin), enzymes
alpha-glucosidase from Saccharomyces cerevisiae, substrates
(4-nitrophenyl b-D-glucopyranoside) and were purchased from
Sigma Chemical Co. (USA). The DPP (IV) inhibitor screening assay
kit was obtained from Cayman Chemicals (Catalog no. 700210)
and was stored at 80
C.
Preparation of extract through SWE method
Subcritical water extraction (SWE) was carried out using a batch
system comprising of a SEPAREX piston pump (P200 LGP50,
France), an electrical control panel and an extraction vessel. The
total volume of the high-pressure stainless steel vessel was
50 ml, thus, it was filled with about 2 g of the C. latifolia root
and fruit extract respectively. Similar to what was done in our
previous research work, the independent variables were meas-
ured at optimum conditions which were set at 180
C for 30 min
of extraction time16. All other parameters were held constant,
comprising of an extraction pressure of 100 bar, the flow rate of
0.5 ml/min, and water to the solvent ratio of 1:10 w/v. After
extraction, the extraction vessel was cooled and depressurised.
The obtained extracts were then concentrated by drying in a
freeze dryer to remove any excess water completely. The pure
extract was then stored in a dark place at 4
C for fur-
ther analysis.
Measurement of enzymatic inhibitory activity
a-Glucosidase inhibitory assay
The a-glucosidase inhibition activity of the sample was deter-
mined following the procedure described with minor modifica-
tions17. A 40-lL aliquot of a-glucosidase solution (1 U/mL) is
added to 1lLof sample solution (500 lg/mL in DMSO) and 69 lL
of 0.1 M sodium phosphate buffer. After 15 min incubation at
37
C, 40 lL of substrate solution (5 mM p-nitrophenyl a-D- gluco-
pyranoside) is added to the reaction mixture and incubated at
37
C for 30 min. Then, the reaction is terminated by adding
150 lL of 0.1 M Na2CO3. The yellow product can be determined at
absorbance 405 nm, using a plate reader (Biotek Synergy H1
Multi-Mode Reader, Winooski, VT, USA). The percentage of inhib-
ition is calculated as follows:
% inhibition ¼ ð1  Asample =Acontrol Þ  100
Reference acarbose also tested as a positive control in this
assay. All samples were analysed in triplicate.
DPP (IV) inhibitory assay
The inhibition of DPP (IV) was conducted by following DPP (IV)
screening assay kit by Cayman Chemical (Michigan, USA), (Item
no: 700210). The positive control inhibitor, 30 ml of sitagliptin and
sample was made in varying concentrations (500–8000 mg/ml) in
an assay buffer was added into10 ml of DPP (IV) enzyme solution.
Then, the solutions were incubated for about 15 min at 37
C. A
50 ml of the substrate was added into the mixture and was incu-
bated for 30 min at 37
C. The fluorescence reading was measured
by setting excitation wavelength at 355 nm, emission wavelength
at 455 nm and gain setting at 80. The percentage of inhibition

was calculated as follows:
% inhibition ¼ ð1  Asample =A100%
initial activity Þ  100
Where 100% initial activity is formed by the combination of
assay buffer, DPP (IV) and distilled water. Sitagliptin is used as a
positive control in this assay. All samples are analysed in triplicate.
Liquid chromatography-mass spectrometry (LC-MS) analysis
LC-MS analysis was performed using MS-Q-TOF mass spectrometer
(Agilent Technologies, Santa Clara, USA) coupled to an HPLC sys-
tem (Agilent Technologies, Santa Clara, USA). A C18 column
(125  2 mm i.d., 5 lm particle size) was used. The mobile phase
was composed of water (A, 6% acetic acid) in acetonitrile (B, 1%
acetic acid), the gradient is as follows: 2–5.00 min; 100% A,
5–10 min 60% A, 10–15 min 50% A, 15–20 min 20%. The sample
injection volume was 10 ll and the mobile phase flow rate was
0.5 ml/min. The column of chromatography was kept at 30
C.
Based on the available mass spectra found in the literature libra-
ries, the identified recognised active compounds are signifi-
cantly identified.
In vitro studies
Cell culture
All the cells including 3T3-L1 preadipocyte cell, L6 muscle cell,
and BRIN BD11 pancreatic cells were obtained from Dr. Muhajir
Hamid, Animal Cell Culture Laboratory, Faculty of Biotechnology
and Biomolecular Sciences, Universiti Putra Malaysia. BRIN BD11
cells were cultured in RPMI growth medium supplemented with
10% (v/v) foetal calf serum (FBS, Gibco/BRL), 100 mg mL  1 peni-
cillin and 10 mg mL  1 streptomycin (Gibco/BRL) at 37
C in 5%
CO2. Cells were passaged every 5–7 days. L6 muscle cell and 3T3
adipose cell is maintained in DMEM supplemented with 10% inac-
tivated Foetal Bovine Serum (FBS), penicillin (100 IU/ml) and
streptomycin (100 g/ml) in a humidified atmosphere of 5% CO2 at
37
C until confluent. The passage number of cell used was
between 20 to 30.
Cell cytotoxicity by MTT assay
Cell viability was assessed by MTT colorimetric assay as reported
with minor modifications18. Cells were seeded at a density of
1  105 cells/well in a 96-well plate and allowed to attach for 24.
The attached cells were then exposed to the test samples with
varying concentrations for a 24 h challenge. Subsequently, 20 lL
of MTT (5 mg/mL) was added and further incubated for another
4 h period. Following removal of excess MTT and PBS rinse, 200 lL
of DMSO was added to dissolve the formazan crystals formed. The
purple formazan coloured product was quantified at 570 nm,
using a plate reader (Biotek Synergy H1 Multi-Mode Reader,
Winooski, VT, USA). In this assay, 1% of DMSO was employed as
vehicle control and Triton X-100 (1.0%; v/v) in DMEM were being
employed as a positive control. The cell viability was expressed as
a fraction of viable cells relative to vehicle control cultures. The
IC20 values and IC50 values, indicating the respective concentration
of test sample producing 20 and 50% inhibition in growth, were
calculated using Graphpad Prism 7.0 (San Diego, California, USA).
3T3–L1 pre-adipocyte differentiation
Differentiation of 3T3-L1 cells was performed following the guide-
line provided by American Type Culture Collection (ATCC)
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 111
Technical Bulletin No. 9 (ATCC, 2011). The cells were first seeded
into 24-well culture plate at a density of 3  104 cells/well and fur-
ther incubated until 100% confluence. The differentiation process
(Day 1) was initiated by incubating the cells in differentiation
medium comprising of DMEM with 10% FBS, 50 lg/mL gentami-
cin, 1.0 mM dexamethasone, 0.5 mM IBMX and 1.0 mg/mL insulin.
Fresh differentiation medium was supplied after every 48 h of
incubation. On day 5, the differentiation medium was replaced
with an adipocyte maintenance medium consisting of DMEM with
10% FBS and 1.0 mg/mL insulin. The replacement of fresh medium
after every 48 h incubation period proceeded until day 12. Control
cultures were treated with either 0.1% DMSO (20 mL) or rosiglita-
zone (5–20 mM).
2-Nbdg uptake assay
Stimulated glucose uptake activity was performed using fluores-
cently-labelled 2 NBDG glucose analog based on modified proto-
col19. The protocol was followed based on the 2-NBDG uptake
cell-based assay kit (Item No: 600470) Cayman Chemical
(Michigan, USA). Glucose uptake activity of test drugs is deter-
mined in L6 and 3T3-L1 cells on 96-well clear bottom black fluor-
escence plates (Thermo Scientific, Pittsburgh, PA, USA). In brief,
the 24 h cell cultures with 70–80% confluency are allowed to dif-
ferentiate by maintaining in DMEM with 2% FBS for 4–6 days. The
extent of differentiation is established by observing the multinu-
cleation of cells. The differentiated cells are serum-starved over-
night and at the time of the experiment, cells are washed with
HEPES buffered Krebs Ringer Phosphate solution (KRP buffer) once
and incubated with KRP buffer with 0.1% BSA for 30 min at 37
C.
Cells are treated with different non-toxic concentrations of test
sample (1.5–0.0468 mg/mL) and (1.0–0.062 mg/mL) for 3T3-L1 and
L6 cell respectively for 60 min along with negative controls in glu-
cose-free culture medium at 37
C. At 10 min before the end of
the treatment, 2-NBDG was added to a final concentration of
200 lg/mL in a glucose-free medium. At the end of treatment, the
plate was centrifuged for 5 min at 400  g at room temperature.
The supernatant was aspirated and 200 lL of cell-based assay buf-
fer was added to each well. The plate was centrifuged for 5 min at
400  g at room temperature. The supernatant was aspirated and
100 lL of cell-based assay buffer was added to each well. The 2-
NBDG taken up by cells can be detected with a fluorescent read-
ing by (excitation/emission ¼ 485/535 nm) by using Biotek
Synergy H1 Multi-Mode Reader (Winooski, VT, USA). Three inde-
pendent experimental values in duplicates are taken to determine
the percentage of 2-NBDG uptake over controls.
Insulin secretion assay
Insulin release was determined using the monolayer of BRIN-BD11
clonal pancreatic cells. BRIN-BD11 cells were grown in RPMI1640
tissue culture medium containing 11.1 mmol glucose/l, 10%foetal
calf serum and antibiotics (50,000 IU penicillin-streptomycin/l), and
maintained at 37
C in an atmosphere of 5% CO2 and 95% air.
Then, 20 h prior to acute experiments, cells were harvested and
seeded in 24-well plates at a density of 1  105 cells/well.
Following overnight attachment, the culture medium was
removed and cells were preincubated for 40 min at 37
C with
1 ml of Krebs ringer bicarbonate (KRB) buffer supplemented with
1.1 mM glucose and 1% bovine serum albumin. Samples were
immediately done for subsequent insulin radioimmunoassay and
were tested by using the Human Insulin Elisa Kit by Kono Biotech
(Zhejiang, China).
112 N. A. ZABIDI ET AL.

Molecular docking Acarbose(control)

100 a
Preparation of ligand Root
The set of ligand molecules studied in this work were obtained
80 Fruit
b a
from the National Centre for Biotechnology Information (NCBI)
PubChem database (http://PubChem.ncbi.nlm.nih.gov). The list of

% inhibition
the ligands present in the extract that initially used are phlorizin 60 b
(CID 6072), berberine (CID:2353), dimethylcaffeic acid (CID: c
717531), monobenzone (CID: 7638), mundulone (CID:4587968), 40 a
d a c a a
pomiferin (CID: 4871), scandenin (CID: 54676535), acarbose (CID: c
41774), rosiglitazone (CID: 77999) and sitagliptin (CID: 4369359). 20
These molecules were downloaded in Structure Data File (SDF)
format and converted to Protein Data Bank (PDB) format. 0

5
25
5
25

2.
62

1.
31

0.
0.
Preparation of receptor
The crystal structure of a-glucosidase (PDB ID: 4J5T), DPP (IV) com- Concentration (mg/ml)
plex enzyme (PDB ID: 2P8S) and insulin receptor (IR) (PDB ID: Figure 1. a-Glucosidase inhibitory effects of the extracts and the acarbose refer-
1IR3) was retrieved from Protein Data Bank (http://www.rcsb.org/ ence as a positive control. Values are expressed as the mean ± SD (n ¼ 3).
pdb). These molecules were downloaded in Protein Data Bank Different letters indicate significant differences (p < 0.05) within the same sam-
ple/control as increasing the concentration.
(PDB) format and the water molecules, as well as other heteroa-
toms, were omitted from the complex protein molecule.
100 Fruit a
a b
Root
Docking 80 Sitagliptin (Drugs) a c
Molecular docking studies were carried out using AutoDock pro-

% inhibition
gram to study the binding within the active site of each of the 60
enzymes. The binding conformation was visualised using PyMOL. c
a b
The grid size was set to 28  28  30 xyz points andgrid centre 40
was designated at dimensions (x¼ 14.748, 16.602, 48.062, a b c
y¼ 27.504, 49.805, 51.582, z¼ 1.293, 21.889, 34.659, for 4J5T, 20
b
1HNY and 2P8S respectively. The grid centre was placed in the b c
active site pocket centre. The grid boxes included the entire bind- 0
ing site of the enzyme and provided enough space for the ligand 00

00

00

00
0
50

10

20

40

translational and rotational walk. A scoring grid is calculated from
the ligand structure to minimise the computation time. After the
complete execution of AutoDock, various conformations of the lig-
and in a complex with the receptor were obtained, which were
finally ranked based on binding energy. The results of the inter-
action were analysed using a Protein-Ligand Interaction Profiler
(PLIP) server (http://plip.biotec.tu-dresden.de/plip-web/plip/index).
Results thus obtained were expressed in terms of free energy
(kcal/mol) of protein-ligand binding.
Statistical analysis
The data were expressed as mean ± SD values. Prism7 was used
for statistical analysis. The cut off for significant variation was set
at p < 0.05 using Duncan’s test.
Results
a-Glucosidase inhibitory activity
The percent of a-glucosidase inhibition activity of C. latifolia root
and fruit extracts was plotted with the function of concentration
in comparison with acarbose that acts as a positive control as
shown in Figure 1, where the concentration of the sample is rang-
ing from 0.3 to 2.5 mg/ml. The result indicates that root extracts
have significantly more potential for inhibiting these enzymes
which inhibit above 50% on the enzyme compared to the fruit
extracts. The highest concentration of roots extract (2.5 mg/ml)
inhibited the maximum percentage inhibition of a-glucosidase
was nearly 56%. Meanwhile, fruit extracts have recorded the
80
Concentration (μg/ml)
Figure 2. DPP (IV) inhibitory effects of the extracts and sitagliptin reference as a
positive control. Values are expressed as the mean ± SD (n ¼ 3). The different let-
ter indicates there is a significant difference by comparing in each sample/drug
under different concentrations (p < 0.05) according to ANOVA and Tukey’s tests.
maximum inhibition activity with 22% at the maximum extract
concentration (2.5 mg/ml) and for fruits extracts, it notified there
was no significant difference as the concentration increased from
0.3125–0.625 mg/ml and after that, as increasing the concentra-
tion, there was significant difference reported. Even though fruit
extracts give a much lower inhibition value as compared with
root, it also showing good inhibition with the a-glucosidase
enzyme. Acarbose appeared to be about 2-fold more active than
root extract and 5 times more active that fruit extract. We also
determined the half-maximum inhibitory concentration (IC50) for
each fraction to quantify the inhibitory potential of the extracts,
resulting in a 50% suppression of the original enzyme activity.,
where root extract was shown as a good inhibitor for a a-glucosi-
dase enzyme with IC50 2.72 mg/ml while the fruit extract recorded
with IC50 3.87 mg/ml.
DPP (IV) inhibitory activity
The inhibitory potential of DPP (IV) by root and fruit of C. latifolia
that also being compared with marketed synthetic drugs,sitaglip-
tin which was used as a positive control in this assay was shown
in Figure 2. The test concentration used was in the range from
 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 113

250 to 3000 lg/ml. From the results, C. latifolia root extracts had
recorded an inhibitory potential of DPP (IV) from 7.02 ± 0.52% to
66.15 ± 4.09% while fruit extract had inhibitory potential from
2.69 ± 0.25% to 42.79 ± 1.47%. DPP (IV) inhibitory potential of
standard drugs, sitagliptin was found to be from 17.94 ± 4.73% to
86.96 ± 1.60%. The maximum inhibition that was achieved for root,
fruits, and drugs at the highest concentration, 3 mg/ml were
66.15, 42.79, and 86.96% respectively. The results also showed
that as increasing the extract’s concentration, the percentage of
inhibition was also increased. There was a significant difference
for the sample extracts and drugs, as were observed in all tested
concentrations.
Screening of active compound using LC-MS analysis
A total 120 of compounds including known and unknown com-
pounds were identified by LC-MS in the negative ion mode. Based
on the results, peaks were labelled according to the order of their
retention time, absorption wavelengths, m/z values, and tentative
identification were aided by the existing literature.In particular,
these compounds were phenolic (monobenzone) and identifica-
tion as phenolic from benzene derivative (hydroquinone), flavon-
oid glycoside (phlorizin), prenylflavonoids (pomiferin), isoflavonoid
(mundulone and scandenin) and in particular cinnamic acid
derivative (dimethyl caffeic acid). The representative chromato-
grams in the screening of C. latifolia root and fruit extracts shown
in Figure 3 and Table 1. In the analysis of root extract, the com-
pound that was detected were as follows: phlorizin (m/z 477),
scandenin (m/z 433), mundulone (m/z 433, 867), hydroquinone
(m/z 109), dimethylcaffeic acid (m/z 683), hordatine A (m/z
532, 549, 565). Meanwhile, the compound that was detected for
the fruit extract were as follows: berberine (m/z 1004), pomiferin
(m/z 447), hordatine A (m/z 549), monobenzone (m/z 399), frangu-
lin B (m/z 401), and robustine (m/z 183, 195, 207, 217, 221, 239).

Figure 3. LC-MS chromatogram screening of C. Latifolia. (A) root extract and (B) fruit Extract. Peaks: 1, phlorizin; 2, scandenin; 3, mundulone; 4, hydroquinone; 5,dime-
thylcaffeic acid; 6, hordatine A; 7, berberine; 8, pomiferin; 9, hordatine A; 10, monobenzone; 11, frangulin B; 12, robustine.

Table 1. LC-MS analysis of C. latifolia.

Sample Peak no. Retention time (min) Compound Formula Fragment ions
Root extract 1 5.185 Phlorizin C21 H24 O10 m/z of 477
2 6.589 Scandenin C26 H26 O6 m/z of 433
3 6.725 Mundulone C26 H26 O6 m/z 433,867
4 7.529 Hydroquinone C6 H6 O2 m/z 109
5 8.447 Dimethylcafeic acid C11 H12 O4 m/z 683
Fruit extract 6 10.74 Hordatine A C28 H38 N8 O4 m/z 532,549,565
7 2.436 Berberine C20 H17 NO m/z 1004
8 5.334 Pomiferin C25 H24 O6 m/z 447
9 9.732 Hordatine A C28 H38 N8 O4 m/z 549
10 12.962 Monobenzone C13 H12 O2 m/z 399
11 13.698 Frangulin B C20 H18 O9 m/z 401
12 15.95 Robustine C12 H9 N O3 m/z 183,195,207, 217,221,239
114 N. A. ZABIDI ET AL.

Table 2. IC20 and IC50 values of C. latifolia root and fruit extract on 3T3-L1 preadipocytes, L6 muscle cell, and BRIN-BD11 pancreatic cell.
3T3-L1 L6 BRIN-BD11
Extracts IC20 (lg/ml) IC50 (lg/ml) IC20 (lg/ml) IC50 (lg/ml) IC20 (lg/ml) IC50 (lg/ml)
Root 153.21 ± 9.65 561.42 ± 6.22 605.53 ± 12.33 982.11 ± 5.53 681.87 ± 15.42 927.42 ± 5.81
Fruit 295.67 ± 7.43 495.67 ± 11.31 625.54 ± 21.43 893.41 ± 6.67 649.82 ± 7.41 1249.82 ± 15.34
Data are expressed as mean ± standard deviation (n ¼ 6).

In vitro studies (A) 100

Root
Cell viability assessment in the presence of C. latifolia extract **** ****

2-NBDG Glucose uptake (%)

The effect of root and fruit extracts on a mechanism in the anti- Fruit
80
diabetic pathway through glucose uptake assay and insulin secre-
tion were investigate by MTT assay. The ability of the cells to
60 *
survive a toxic insult has been the basis of the cell viability assess- *** ****
ment. Cell viability assessment was undergone throughout varied **** ****
extracts concentrations (0.25 mg/ml-2 mg/ml) that depends on the 40
toxicity of the extracts in each of the cells. The comparison of IC20 **** **** ****
and IC50 was summarised in Table 2. From the results, the toxicity 20
for roots had recorded IC20 (153.21 ± 9.65 lg/ml) and IC50
(561.42 ± 6.22 lg/ml), a little bit higher than fruits extract with IC20 0
(295.67 ± 7.43 lg/ml) and IC50 (495.67 ± 11.31 lg/ml) in 3T3–L1 cell.

lin

00
8

0
47

94

18

37

75
With the treatment on L6 cells, roots had recorded IC20

10
su
(605.53 ± 12.33 lg/ml), IC50 (982.11 ± 5.53 lg/ml), while fruits

In
extract with IC20 (625.54 ± 21.43 lg/ml), IC50 (893.41 ± 6.67 lg/ml). Concentration (μg/ml)
The results for in the cell viability in BRIN-BD11 cell was achieved
*
with root extracts recorded IC20 (681.87 ± 15.42 lg/ml), IC50 (B) 100 Root
(927.42 ± 5.81 lg/ml), while fruits extract with IC20 2-NBDG Glucose uptake (%) Fruit
(649.82 ± 7.41 lg/ml), IC50 (1249.82 ± 15.34 lg/ml). Generally, the 80
dose of each treatment in the extracts is partially based on the
60 *
MTT assay where the safe concentration for the cell to be induced
with tested extract and fractions. Thus, concentration up to ****
40
1000 lg/ml was employed in a further assay for in vitro studies.
**** ****
20 ****

Glucose uptake activity of C. latifolia extract in 3T3-L1 adipocyte 0

and L6 muscle cell
lin

00
.5

0
12

25

50
62

10
su

The result given by the uptake of glucose showed from the activ-
In

ity in 3T3–L1 and L6 cell line were shown in Figure 4. The result
was compared with insulin (injectable antidiabetic) at a fixed con-
centration of 250 lg/ml. The given results show that the root
extract achieved a higher percentage in both cell lines with 85
and 67% for L6 and 3T3-L1 cells respectively as compared with
fruit extracts. In 3T3–L1 adipocyte cells, we found that the root
extract of C. latifolia showed an increase of glucose uptake 750
and 1000 lg/mL while fruit extract recorded an increment of glu-
cose uptake at concentration 1000 lg/ml when compared to the
control insulin. However, based on results, we can observe that in
order to get higher glucose uptake as same as insulin, the concen-
tration of the extract was 4 fold higher than the concentration of
insulin. The results revealed that there was a significant difference
(p < 0.0001) increases in the uptake reaction were observed with
the incubation with respective 47, 94, and 1000 mg/ml of root and
47, 94, 188, 375, and 1000 mg/ml. On the other hand, in L6 myo-
blast cells, we found that the root extract of C. latifolia showed an
increase of glucose uptake 1000 lg/ml while fruit extract recorded
a slight lower in glucose uptake at concentration 1000 lg/ml
when compared to the control insulin. However, based on results,
we can observe that in order to get higher glucose uptake as
same as insulin, the concentration of the extract was 4-fold higher
than the concentration of insulin that contributes to the above
80% uptake of glucose by L6 cell. The results revealed that there
was a significant difference (p < 0.0001) increases in the uptake
Concentration (μg/ml)
Figure 4. Percentage of glucose uptake after treatment with various concentra-
tion (A) 3T3-L1 adipocyte cell; (B) differentiated L6 muscle cell. Values are
expressed as the mean ± SD (n ¼ 6). p < 0.05, p < 0.01, p < 0.001 and
p < 0.0001 compared to the control.
reaction were observed with the incubation with respective 62.5
and 125 mg/mL for both of the extracts.
Insulin secretion activity of C. latifolia extract in BRIN-BD11 pan-
creas cell line
The secretion of insulin assay was conducted by using the BRIN-
BD11 cell line, which is a clonal insulin-secreting cell that responds
to a variety of insulin-secreting pharmacological modulators. This
cell line is mainly used to examine the C. latifolia extracts upon the
insulin secretion mechanism assay. Insulin release by BRIN-BD11
cells was significantly increased in a dose-dependent manner for
both extracts as the sample concentration (0.125–1 mg/ml) (Figure
5). In this mechanism of the assay, glibenclamide (oral antidiabetic
drugs) acted as positive control. However, the concentration of gli-
benclamide was standardises at 0.5 mg/ml. Root extract subse-
quently increase with 5.02 ± 1.42 lg/ml, 7.32 ± 2.23 lg/ml,
11.48 ± 0.51 lg/ml, 13.74 ± 0.57 lg/ml at concentration 125, 250,
 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 115

Fruit 500, 1000 lg/ml, respectively. On the other hand, the secretion of
15 Root insulin for fruit extract also subsequently increase with 5.16 ± 0.79,
5.96 ± 1.29, 7.91 ± 0.79, 11.06 ± 0.35 lg/ml at concentration 125,
250, 500, 1000 lg/ml, respectively. Even though root extract shows
Insulin secretion ((μg/L)

10 * a slightly higher secretion of insulin as compared with glibencla-

** mide that recorded with 12.88 ± 0.79 lg/ml at a concentration
*** (500 lg/ml), but the extract concentration was 2-fold higher than
*** **
glibenclamide. However, both of the extracts showed a positive
5
result by having an increased insulin release that comparable with
glibenclamide results.

0
Molecular docking
e

00
5

0
id

12

25

50

10
m
la

Concentration (μg/ml) Molecular docking studies were used in order to investigate the
nc
be

binding confirmation to the selected compound. At the beginning

ly

of the process for docking, water molecules have been omitted in

G

Figure 5. Insulin secretion assay for root and fruit of C. latifolia extracts. Values
are expressed as the mean ± SD (n ¼ 5). p < 0.05, p < 0.01, p < 0.001 and
p < 0.0001 compared to the control.
order to explore additional docking posses between compounds
without the possible in the direct hydrogen bond formed. The
molecular structure of selected ligands and active pocket sites for
each of the receptors, 4J5T (a-glucosidase), 2P8S (DPP IV), and
1IR3 (insulin receptor, IR) were displayed in Figures 6 and 7,

Figure 6. Compounds from SWE of C. latifolia with potential antidiabetic activity selected for in silico studies with selected anti-diabetic drugs.
116 N. A. ZABIDI ET AL.
Figure 7. Active site in each targeted protein(A) Structure of a-glucosidase enzyme surface (green) with an active pocket site (red) and active site of the protein. (B)
Structure of DPP (IV) enzyme surface (blue) with the active pocket site (pink) and active site of the protein.(C) Structure of Insulin receptor (green) with the active
pocket site (pink) and active site of the protein.
respectively. In order to explore binding energies (kcal/mol) and
basis molecular interaction, all the ligands were docked with each
of the proteins. The binding energy was indicative of the ligand’s
contribution and flexibility for the targeted protein. In terms of
binding energy, the more negative the value of the standard free
energy charge, the better the binding affinity of the ligand with
the receptor, hence, the more stable the complex20. Therefore, the
lowest binding energy scores represent the best protein-ligand
binding stability compared to the highest energy score. The dock-
ing analysis with each of the receptor generate the binding ener-
gies on each ligands as follows: phlorizin (-8.2, 10.9, 7.0 kcal/
mol), scandenin (-8.0, 9.3, 6.2 kcal/mol), pomiferin (-8.0, 9.6,
6.6 kcal/mol), berberine (-7.9, 8.9, -6.3 kcal/mol), monobenzone
(-7.2, 7.4, 5.4 kcal/mol), mundulone (-6.1, 9.3, 6.9 kcal/mol),
dimethycaffeic acid (-6.0, 7.1, 5.6 kcal/mol) for 4J5T, 2P8S and
1IR3, respectively. Meanwhile, the binding energies that were ana-
lysed for reference drugs acarbose (-7.4 kcal/mol), sitagtiptin
(-8.8 kcal/mol), and insulin (-7.9 kcal/mol). The binding energies on
each of the ligands were shown in Figure 8. The molecular inter-
action involved such as the number of hydrogen bond (H-bond),
hydrophobic bonds, salt bridges, and p-staking as showed in
Table 3. All details of the interaction including amino acid residue
involved, bond length (Å), angle (o) and protein donor are given
in appendices.
From the docking results with receptor 4J5T, among all the
tested compounds, phlorizin was analysedH-bond interactions
with Trp391, Asp392, Asp569 and Trp710, a hydrophobic inter-
action with Phe385, p-staking with Tyr709 and also salt bridges
with Arg428. The amino acid residue of H-bond (Asp392 and
Arg428), along with hydrophobic bonds, Leu563 were observed
forming interaction with compound pomiferin. Scandenin and
mundulone had a similar no of H-bond with Tyr709 and Arg428
respectively. Meanwhile, dimethylcaffeic acid, berberine and
monobenzone have reported not forming any H-bond but

dimethylcaffeic acid was supported with hydrophobic bonds
(Asp568, Tyr709), a p-staking (Trp710) and a salt bridges (His561),
berberine was supported with a p-staking (Tyr709) and a salt
bridges (Asp568), and monobenzone was notified forming with
p-staking interaction (Phe385, Phe389, Trp710). In the docking
results with receptor 2P8S, phlorizin displayed H-bond interaction
with Arg125, Asp545, Asp454, Gln553, Lys554, Trp629 amino acid
residues and was stabilised with a hydrophobic bond (Tyr547) and
p-staking (Tyr547). Pomiferin was notified forming H-bond
(Asp556, Asp560), hydrophobic bond (Trp629) and mundulone
were forming H-bond (Ser630, His740) along with hydrophobic
bond (Trp629). Meanwhile, berberine and dimethylcaffeic acid
formed H-bond with Gln553, Ser630 residues with a hydrophobic
bond (Tyr547). Furthermore, monobenzone was forming a H-bond
(Ser630) and a p-staking (Tyr547) while scandenin was reported
with no H-bond formed but was supported with a hydrophobic
bond (Trp629) along with Tyr547 residues for p-staking inter-
action. For docking analysis with 1IR3, phlorizin showed that for-
mation of six H-bond (Glu1001, Gln1004, Lys1085, Ser1086,
Asn1097), scandenin and mundulone with four H-bond (Arg1000,
Arg1026, Ala1080) and (Ser1086, Asp1156, Thr1160) respectively.
2 through molecular docking studies that reveal the binding inter-
Binding energy (kcal/mol)
0 α -glucosidase
−2
−4
−6
−8
−10
−12
phloridzin dimethylcaffeic
acid
pomiferin
Figure 8. Binding energy between targeted protein and ligand. DPP (IV):
Dipeptidyl Peptidase IV; IR: insulin receptor.
Table 3. Molecular docking analysis.
Enzyme 1 (4J5T)
Compound 1 (acarbose)
Compound 2(phlorizin)
Compound 3 (pomiferin)
Compound 4 (scandenin)
Compound 5 (mundulone)
Compound 6 (monobenzone)
Compound 7 (dimethylcaffeic acid)
Compound 8 (berberine)
Enzyme 2 (2P8S)
Compound 1(phlorizin)
Compound 2 (mundulone)
Compound 3 (dimethylcaffeic acid)
Compound 4 (sitagliptin)
Compound 5 (pomiferin)
Compound 6 (berberine)
Compound 7(monobenzone)
Compound 8 (scandenin)
Enzyme 3 (1IR3)
Compound 1(phlorizin)
Compound 2(mundulone)
Compound 3 (scandenin)
Compound 4 (pomiferin)
Compound 5(dimethylcaffeic acid)
Compound 6 (Insulin)
Compound 7 (berberine)
Compound 8(monobenzone)
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 117
Besides, other compounds such as pomiferin and dimethylcaffeic
acid formed three H-bond (Glu1047, Gly1152, Met1153) and
(His1081, Asp1083, Ser1086) residues respectively. Berberine
showed interaction with one H-bond (Gln1004) while monoben-
zone was analysed with none of H-bond formed but having a
p-staking interaction (Tyr10). To sum up, we observe phlorizin
forms the most stable binding in most of the receptor especially
with DPP (IV) and was selected as a best-docked molecule as
shown in Figure 9.
Discussions
The coordinated function of several different tissues and organs
depends on the optimal regulation of blood glucose. These
include the gut, which is responsible for digestion and absorption
of carbohydrates and pancreatic b-cells, which are responsible for
the precise secretion of insulin. In the present study, the effect of
antidiabetic activity were examined including the inhibitory poten-
tial of a-glucosidase, DPP (IV) enzyme, glucose uptake and secre-
tion of insulin from the C. latifolia root and fruit extracts that were
collected by using subcritical water extraction (SWE) technique.
This research study also completed by a computational approach
DPP (IV) action and other bonds that formed between the proteins with
IR
the receptor involved. Many earlier studies have revealed the role
of plant extracts as a natural inhibitor that has a less adverse
effect and is capable of reducing hyperglycaemia particularly in
the inhibitory activity of the a-glucosidase enzyme8. From the
results, the inhibitory activity on both of the extracts shows expli-
cated a remarkable inhibition on the a-glucosidase enzyme by
revealing the percentage inhibition by root extracts is closer with
the percentage of inhibition by marketed anti-diabetic drugs, acar-
bose. Previously, the findings of the current study are in a good
monobenzone reference agreement with other research works indicate that SWE extracts
mundulone scandenin are capable of influencing a-glucosidase inhibitory capacity21.
Besides, the presence of several phytochemicals such as flavo-
noids, phenolic with a greater number of hydroxyl groups that
H-bond Hydrophobic bonds Salt bridges p-Staking
9 1 – –
5 1 1 1
3 2
1 5 – –
1 2 – –
– – – 3
– 2 1 1
– – 1 1
9 1 – 1
3 1 – –
2 1 1 1
2 – – 1
2 1 – –
2 – – –
1 – – 1
– 2 2
6 – – –
4 1 – –
4 – – –
3 – – –
3 – – 1
1 – 4 1
1 – – –
– – – 1
118 N. A. ZABIDI ET AL.
Figure 9. Best docked of complex molecule. (A) a-glucosidase (B) DPP (IV) and
(C) IR. Blue structures indicate the enzyme/protein, the blue line indicates the
hydrogen bonds formed between the residues, and the orange structures indicate
the ligands involved respectively for both diagrams. The green line indicates the
p-staking interactions and yellow lines indicate the formed salt bridge.
present in the compounds would have a significant inhibitory
effect that boasts a greater inhibitory effect towards the a-glucosi-
dase enzyme22. Significantly, the results on the inhibitory activity
increased in conjunction with the increased of the concentration
of the extract23. Previously, it has been reported that some studies
shows a positive relationship between total polyphenol content
and its ability to inhibit a-glucosidase24. Besides, the results given
by using subcritical water extraction has a remarkable activity in
inhibition of a-glucosidase. By applying hot extraction technique,
the inhibition of a-glucosidase on Meriandra bengalensis has
revealed that the inhibitory activity from water extracts achieved
high inhibitory activity as compared with other organic solvents24.
This can be related with the principal of water under subcritical
state, where the polarity of water decrease as increasing the water
temperature. This will causes the decrease of dielectric constant of
water originally at ambient temperature is e ¼ 80 significantly
reduced to (e ¼ 27 at 250
C), which was pretty closed to the per-
mittivity of ethanol (e ¼ 25) or methanol (e ¼ 33)25. Hence, these
factors reducing the results of the gap produced between the
solvent and SWE extraction methods. Therefore, the findings
revealed that the extracts of C. latifolia from SWE extraction have
potential in inhibiting a-glucosidase activity, which works as one
of the natural inhibitors for a-glucosidase that associated
with T2DM.
With insulin resistance being central in type 2 diabetes patho-
genesis, a new alternative in the inhibition of DPP (IV) has pro-
posed in order to stimulate the release of insulin. From the
inhibitory activities of the extracts with DPP (IV), the results
revealed that both of the extracts displayed a good inhibitory
potential by having an increment in the percent of inhibition for
the DPP (IV) enzyme.
Besides, it can be noticed that C. latifolia root extract acted as
a better inhibitor as compared with the fruit extracts. This is pos-
sibly due to the inhibition from the bioactive compound, contain-
ing mixtures of several phenolics and flavonoids that contained in
the root extracts that may acts as an inhibitor for the inhibition of
the DPP (IV) enzyme26. In addition, some bioactive compounds
containing in crude extracts can exert their properties in DPP (IV)
inhibition because previous studies reported that subclasses in
phenolic, flavonoid, terpenoids, and peptides were the most pre-
dominant compound as DPP (IV) inhibitors27. Whereby, the inhib-
ition activity by the fruit extracts is probably due to the berberis
compound that was present in the extract, where the activity of
berberis is quite similar to DPP (IV) inhibitor, benzoquinolizines
and it was demonstrated berberine significantly reduced the fast-
ing blood glucose (FBG), lowered blood glucose level through
increasing insulin receptor expression in T2DM28. The viability of
the cell was assessed through MTT assay and the results were
employed in determining the IC20 and IC50 of the cell growth.
Fruit extracts proposed a lower toxicity effect at IC50 for 3T3–L1,
L6, and BRIN-BD11 cell lines as compared with the root extract.
Moreover, less toxicity of the sample was probably by applying
the optimum condition of SWE that leads to high polarity and
reduced toxicity. The sample’s concentration lower than 1 mg/ml
was considered as a safe concentration to pursue in vitro assay
activities in the glucose uptake and insulin secretion assays.
Medicinal plants enhance the glucose uptake by GLUT4 trans-
location and were proven by the in vitro glucose model. The L6
and 3T3-L1 cell lines are the best-characterized cellular model ori-
gin to study glucose uptake and GLUT4 translocation. The selec-
tion of cell line was based on the involved in the mechanism of
action in the uptake of glucose where adipose tissue and skeletal
muscle are now well established as a primary target site in glu-
cose metabolism as well as regulation in whole-body glucose
homeostasis and main targets for insulin-stimulated glucose
uptake to control hyperglycaemia29. Skeletal muscle cell acts as a

major primary site that was responsible for postprandial glucose
uptake. In the skeletal muscle, insulin increases glucose uptake by
increasing functional glucose transport molecules in the plasma
membrane30. Furthermore, it is the most abundant tissue in the
whole body and thus, the proper function of skeletal tissue is
important to maintain normal blood glucose levels. In fact, L6 cells
represent a good model for glucose uptake because they have
been used extensively to elucidate the mechanisms of glucose
uptake in muscle, have an intact insulin signalling pathway and
express the insulin-sensitive GLUT431. Based on our findings
showed that root extract displays a better activity with the
increased stimulation of the 2- NBDG uptake in both of the cell
lines as compared with fruit extract. We can observe that the 2-
NBDG uptake increased as the dose concentration of the sample
increase. This is in agreement with previous studies, which have
addressed the increment of 2- NBDG uptake in 3T3-L1 and L6 cell
lines along with the increase in the sample treatments32. The pres-
ence of polyphenols including berberine, flavonoid glycoside
(phlorizin), isoflavonoid (mundulone and scandenin) and cinnamic
acid derivative (dimethyl caffeic acid) that containing in the
extracts, contribute in promoting glucose absorption that can
arise from their synergistic or complementary interactions with
multiple targets in order to exercise their pharmacological behav-
iour. Firstly, berberine is shown to mediate its action by encourag-
ing translocation of GLUT4 and increased activity of AMP-
activated protein kinase (AMPK) in L6 myotubes and mediating
anti-diabetic properties33. Besides, cinnamic acid derivatives pre-
cisely activate proteins phosphoinositide 3-kinase (P13K) pathway
and exert its anti-diabetic effect by substantially enhance the
uptake of glucose34. The finding from previous studies suggests
that the introduction of a hydroxyl group on the benzene ring of
cinnamic acid at the meta- and/or para-position increases the abil-
ity to stimulate glucose uptake in the cells35.
Apparently, the active compounds regulated in the extracts
also contribute to promoting the effects by improving the secre-
tion of insulin. Relatively, the observation made on the in vitro
effect of the extracts on insulin secretion seen in BRIN-BD11 cell
line, clearly establishes that root extracts appear to display a bet-
ter activity in the secretion of insulin as compared with fruit
extracts. The presence of phytochemicals in the extracts that facili-
tate the insulin release on pancreatic cell substantiates our
observed effectiveness. With the presence of cinnamic acid deriva-
tives, this active compounds capable in inducing Ca2þ in pancre-
atic b-cell and this indicates that the stimulating insulin secretion
process by cinnamic acid is due to an increase in Ca2þ flow
through the L-type Ca2þ channels without causing membrane
depolarisation by closing the KATP channels34. The consequent
depolarisation leading to the opening of Ca2þ channels and Ca2þ
intracellular elevation mediating insulin secretory vesicle exocyt-
osis36. These findings were in well correlate with other studies
that reporting the enhancement in promoting insulin release that
being stimulated with the presence of cinnamic acid37.
Consequently, these studies further confirmed with molecular
docking, superimposition of the docked molecule to the receptor
with a-glucosidase, DPP (IV) and insulin receptor (IR) crystal struc-
ture and related interactions. Lower binding energy is equal to
better receptor binding of a ligand. Therefore, the lowest binding
energy scores represent the best protein-ligand binding stability
compared to the highest energy scores. It was revealed that phlor-
izin significantly has the lowest binding energy and the highest
formation of H-bond in all the receptors involved. Practically, the
conformation structure of the ligand with contains more hydroxyl
groups due to the targeted protein happens to be very sensitive
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 119
to the positions of hydroxyl and greatly leading to increase bind-
ing affinities with more tight binding and more potent inhibitory
effects38. The activity of phlorizin was also supported from thepre-
vious studies, which demonstrated the potential of phlorizin that
binds near the active site pocket of the SGL2 protein, which
meant the inhibitor could block the interaction between SGLT2
and glucose, and thus absorption and resulting in the normalisa-
tion of blood glucose9. In the a-glucosidase docking analysis, acar-
bose demonstrated the highest H-bond on the binding with the
receptor. Indeed, acarbose is a well-known antidiabetic drug used
to treat type 2 diabetes by inhibiting a-glucosidase, has been
found to anchor a-glucosidase in the enzyme pocket (domain A)
and to bind to residues like Phe384, Arg387, Trp391, Asp392,
Arg428, Asp569, Tyr709, Glu771 resulting in a powerful inhibition
of a-glucosidase. In the present study, we noticed that hydroxyl
groups on sugar moiety of acarbose are favourable for the ligand
to interact with the amino acid residues of the binding pocket. In
addition, acarbose was observed to bind with the residues like
Asp569 and Glu771 that was fixed at the active site of a-glucosi-
dase. However, interesting findings were also be found with phlor-
izin that forms as the second-highest in the formation of H-bond
followed by pomiferin, scandenin. mundulone that revealed the
potential of phlorizin to bind at one of the pocket active sites of
a-glucosidase, which at residue Asp569 and the binding of these
bonds with the enzyme catalytic site play a major role that
responsible for the inhibitory activity of a-glucosidase.
The binding at the active site plays a vital role in increasing
the binding affinities between ligand-receptor. Phlorizin was found
to form nine hydrogen bonds with DPP (IV) among the seven
compounds, and its binding energy was the highest among the
seven compounds. We observe that the binding of phlorizin form-
ing two H- bonds with active site residues of DPP (IV), Arg125
contribute to the lowest binding energy formed due to the
molecular shape and size of a compound that makes it easier to
penetrate the binding pockets. Meanwhile, compounds such as
monobenzone, mundulone, berberine and dimethylcaffeic acid
were also observed with a quite high binding energy is probably
due to the formation of H-bonds at the active site, residue like
Ser630. We also notified that the majority of the compounds such
as berberine, dimethylcaffeic acid, phlorizin, monobenzone, sande-
nin found to be exclusively involved in the p-staking interaction
with one of DPP (IV) active site, Tyr547 as similar with sitagliptin.
These factors claimed as one of reason on these compounds have
the ability to mimic the binding interaction and found to follow
the same interaction pattern as compared with sitagliptin.
Theoretically, p-staking was analysed as the three top most bind-
ing interaction in protein-ligand binding including H-bonds,
hydrophobic bonds and others that also enhance binding affin-
ity39. It is believed that p-staking is considered as one of the nat-
ural keys in non-covalent interaction and the presence of these
bonds is significant in many biological systems as solitary effects
but also their interplay omnipresent40. Moreover, the docking with
the insulin receptor (1IR3) was also included in these studies.
Residues of amino acids, namely Ser1006, Lys1030, Asp1083,
Met1079, Ala1080, and Glu1077, were the major residues of amino
acids involved in internal ligand interaction and most of the active
components in 1IR3. According to the molecular docking results,
pomiferin and scandenin were the only compounds that were
notified to interact with one of the active sites of 1IR3, which are
Asp1083 and Ala1080 respectively through hydrogen bonding
interaction. By interacting with the active site of IR, these com-
pounds have the potential to act as IR activators therefore, adjust
blood glucose to promote the utilisation of glucose. Besides,
120 N. A. ZABIDI ET AL.
results also showed that for ligands with high-affinity scores were
also influenced by the increase in the number of H-bond formed.
The presence of H-bonds is definitely attributed to form a major
interaction and regarded to be the main driving force of conform-
ational change of the receptor upon ligand binding. To the best
of our knowledge, our present studies were found to be as the
first studies that revealed the molecular docking with the com-
pounds that present in C. latifolia extracts and this works is based
on continuation from our previous studies that involving with
antioxidant activity and screening of compounds by LC-MS ana-
lysis16. Thus, these studies have been pointed out the potential of
phlorizin by inhibiting the activity of a-glucosidase and DPP (IV)
enzyme as the new alternative for antidiabetic drugs that poten-
tially being used in the treatment for type 2 diabetes.
Conclusion
As a conclusion remarks, the results from our current study
showed that the root and fruit extracts of C. latifolia likely to exert
its anti-diabetic properties through inhibitory activity by a-glucosi-
dase that involves in the digestion of carbohydrate mechanism
and DPP (IV) enzyme in intestine that stimulating the secretion of
insulin and reducing glucose level in the bloodstream.
Additionally, both extracts were also observed to enhance in stim-
ulating glucose uptake and insulin secretion. Briefly, the results
also highlighted root extracts producing better antidiabetic activ-
ities as compare with fruit extracts. The effects of phytochemical
compounds that enhance in antidiabetic activity were also being
studied thoroughly by molecular docking that revealed the inter-
esting findings of phlorizin compounds as the promising candi-
dates which dock well with targeted protein and responsible in
inhibitory activities. In fact, the quantification of each of the com-
pound from the results on the screening from LCMS analysis was
still in the progress. However, future studies should address the
molecular mechanisms that may be related to the metabolic path-
way for treating diabetes and isolation of the active constituents
will be interesting to explore and can be considered for develop-
ing into a potent antidiabetic drug.
Acknowledgements
The authors acknowledge the financial support by the Putra
Graduate Initiative Grant (GP-IPS) under vote number 9645100,
Universiti Putra Malaysia (UPM). The authors also would like to
thanks the UPM for providing her financial assistance under the
Graduate Research Fellowship (GRF) program and Institute
Bioscience, Universiti Putra Malaysia (UPM) for providing labora-
tory facilities
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was funding by Putra Graduate Initiative Grant (GP-IPS)
under vote number [9645100], Universiti Putra Malaysia (UPM).
Data availability statement
The data used to support the findings of this study are available
from the corresponding author upon request.
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